Silk fibroin fiber bundles for matrices in tissue engineering

ABSTRACT

The present invention provides a novel silk-fiber-based matrix having a wire-rope geometry for use in producing a ligament or tendon, particularly an anterior cruciate ligament, ex vivo for implantation into a recipient in need thereof. The invention further provides the novel silk-fiber-based matrix which is seeded with pluripotent cells that proliferate and differentiate on the matrix to form a ligament or tendon ex vivo. Also disclosed is a bioengineered ligament comprising the silk-fiber-based matrix seeded with pluripotent cells that proliferate and differentiate on the matrix to form the ligament or tendon. A method for producing a ligament or tendon ex vivo comprising the novel silk-fiber-based matrix is also disclosed.

FIELD OF THE INVENTION

The present invention is directed to a matrix or scaffold used in theproduction of bioengineered tissue, particularly ligaments and tendons.More particularly, the invention relates to a novel silk-fiber-basedmatrix upon which pluripotent cells may be seeded ex vivo and whichproliferate and differentiate thereon into an anterior cruciate ligamentfor implantation into a recipient in need thereof.

BACKGROUND OF THE INVENTION

Every year, hundreds of thousands of Americans sprain, tear, or ruptureligaments and tendons of the knee, elbow, hand, shoulder, wrist and jaw(Langer et al., Science 260: 920-926 (1993)). Of particular importanceis the anterior cruciate ligament of the knee. More than 200,000 peoplein the U.S. alone, will tear or rupture their anterior cruciate ligament(ACL) each year (Albright et al., 1999. Chapter 42-Knee andLeg:Soft-Tissue Trauma. In Orthopaedic Knowledge Update 6. AmericanAcademy of Orthopaedic Surgeons). ). The ACL serves as a primarystabilizer of anterior tibial translation and as a secondary stabilizerof valgus-varus knee angulation, and is often susceptible to rupture ortear resulting from a flexion-rotation-valgus force associated withsports injuries and traffic accidents. Ruptures or tears often result insevere limitations in mobility, pain and discomfort, and the loss of anability to participate in sports and exercise. Failures of the ACL areclassified in three categories: (1) ligamentous (ligament fibers pullapart due to tensile stress), (2) failure at the bone-ligament interfacewithout bone fracture, and (3) failure at the bone-ligament interfacewith bone fracture at the attachment site of bone and ligament. The mostcommon type of ACL failure is the first category, ligamentous.

It is widely known throughout the medical community that the ACL haspoor healing capabilities. Total surgical replacement and reconstructionare required when injury to the ACL involves significant tear orrupture. Four options have been utilized for repair or replacement of adamaged ACL: (1) autografts, (2) allografts, (3) xenografts, and (4)synthetic prostheses (degradable and non-degradable). To date, nosurgical repair procedure has been shown to restore knee functioncompletely, and novel treatment options would likely benefit a largenumber of patients.

The problems associated with the use of synthetic ACL replacements,along with the limited availability of the donor tissue, have motivatedresearch towards the development of functional and biocompatibleequivalents of native tissues. This shift from synthetic tobiologically-based ACL replacements first applied in early studies inwhich collagenous ACL prostheses were prepared as composite structuresconsisting of reconstituted type I collagen fibers in a collagen Imatrix with polymethylmethacrylate bone fixation plugs, and used asanterior cruciate ligament replacement tissues in rabbits (Dunn et al.,Am. J. Sports Medicine 20: 507-515 (1992)). Subsequent studiesincorporated active biological components into the process, such asligament fibroblasts seeded on cross-linked collagen fiber scaffoldsthat were used as ligament analogs (Dunn et al., J. Biomedical MaterialsRes. 29: 1363-1371 (1995); Dunn, M. G., Materials Res. Soc. Bulletin,November: 43-46 (1996)), and suggested that structures approximatingnative ligaments can be generated.

A tendon gap model, based on pre-stressed collagen sutures seeded withmesenchymal stem cells provided improved repair of large tendon defects(Young et al., 1998). Goulet et al. modified the collagen-fibroblastsystem by using ligament fibroblasts in non-cross-linked collagen, withbone anchors to pre-stress the tissue and facilitate surgicalimplantation (Goulet et al., Tendons and Ligaments. In Principles ofTissue Engineering, Ed. R. Lanza, R. Langer, W. Chick. R. G. Landes Co.pp 633-643, R. G. Lanz Co. and Academic Press, Inc., San Diego, Calif.(1997)). Passive tension produced by growing the new ligament in avertical position induced fibroblast elongation and the alignment of thecells and surrounding extracellular matrix.

Silk has been shown to offer new options for the design of biomaterialsand tissue-engineering scaffolds with a wide range of mechanicalproperties (Sofia, S., et al., J. Biol. Mat. Res. 54: 139-148 (2001).For example, the dragline silk from the orb weaving spider, Nephiliaclavipes, has the highest strength of any natural fiber, and rivals themechanical properties of synthetic high performance fibers. Silks alsoresist failure in compression, are stable at high physiologicaltemperatures, and are insoluble in aqueous and organic solvents. Inrecent years, silks have been studied as a model for the study ofstructure-function relationships of fibrous proteins. The manipulationof silk genes, both native and artificial versions, has provided insightinto silk protein expression, assembly, and properties. Thus,biocompatibility, the ability to engineer the materials with specificand impressive mechanical properties, and a diverse range of surfacechemistries for modification or decoration suggests that silk mayprovide an important class of biomaterial. Recent studies by theinventors of the present invention have demonstrated the successfulattachment and growth of fibroblasts on silk films from silkworm silk ofBombyx mori.

Tissue engineering can potentially provide improved clinical options inorthopaedic medicine through the in vitro generation of biologicallybased functional tissues for transplantation at the time of injury ordisease. Further, adult stem cells are becoming increasingly recognizedfor their potential to generate different cell types and therebyfunction effectively in vitro or in vivo in tissue repair. (Sussman, M.Nature 410: 640 (2001). The knee joint geometry and kinematics and theresultant effects on ACL structure must be incorporated into theconstruct design if a tissue engineered ACL generated in vivo is tosuccessfully stabilize the knee and function in vivo. A mismatch in theACL structure-function relationship would result in graft failure.

To date, no human clinical trials have been reported with tissue culturebioengineered anterior cruciate ligaments. This is due to the fact thateach approach has failed to address one or more of the following issues:(1) the lack of a readily available cell or tissue source, (2) theunique structure (e.g., crimp pattern, peripheral helical pattern andisometric fiber organization) of an ACL, and (3) the necessaryremodeling time in vivo for progenitor cells to differentiate and/orautologous cells to infiltrate the graft, thus extending the time apatient must incur a destabilized knee and rehabilitation. Thedevelopment of a matrix for generating more fully functionalbioengineered anterior cruciate ligaments would greatly benefit thespecific field of knee reconstructive surgery, and would also providewider benefits to the overall field of in vitro tissue generation andreplacement surgery.

SUMMARY OF THE INVENTION

The present invention provides a novel silk-fiber-based matrix forproducing ligaments and tendons ex vivo. More specifically, the presentinvention is directed to engineering mechanically and biologicallyfunctional anterior cruciate ligament using a novel silk-fiber-basedmatrix that may be seeded with pluripotent cells, such as bone marrowstromal cells (BMSCs). The mechanically and biologically autologous orallogenic anterior cruciate ligament comprised of the novel matrix andpluripotent cells may be prepared within a bioreactor environment toinduce de novo ligament tissue formation in vitro prior to implantation.Surprisingly, it has now been found that the novel silk-fiber-basedmatrix supports BMSC differentiation towards ligament lineage withoutthe need for directed mechanical stimulation during culture within abioreactor. The inventors believe that mechanical stimulation will serveonly to enhance the differentiation and tissue development process.

The present invention also provides a method for the generation oftissue engineered ACL ex vivo using the novel silk-fiber-based matrixthat comprises the steps of seeding pluripotent stem cells in thesilk-fiber-based matrix, anchoring the seeded matrix by attachment to atleast two anchors, and culturing the cells within the matrix underconditions appropriate for cell growth and regeneration. The culturingstep may comprise the additional step of subjecting the matrix to one ormore mechanical forces via movement of one or both of the attachedanchors. In a preferred embodiment for producing an ACL, pluripotentcells, and more particularly bone marrow stromal cells, are used.Suitable anchor materials comprise any materials to which the matrix canattach (either temporarily or permanently), and which supports ligamentand tendon tissue growth or bone tissue growth at the anchors. Preferredanchor materials include hydroxyappatite, demineralized bone, and bone(allogenic or autologous). Anchor materials may also include titanium,stainless steel, high density polyethylene, Dacron and Teflon, amongstother materials. Goinopra coral which has been treated to convert thecalcium carbonate to calcium phosphate has also been used as an anchormaterial. In a preferred embodiment, the mechanical forces to which thematrix may be subjected mimic mechanical stimuli experienced by ananterior cruciate ligament in vivo. This is accomplished by deliveringthe appropriate combination of tension, compression, torsion and shear,to the matrix.

The bioengineered ligament which is produced according to the presentinvention is advantageously characterized by a cellular orientationand/or matrix crimp pattern in the direction of applied mechanicalforces, and also by the production of ligament and tendon specificmarkers including collagen type I, collagen type III, and fibronectinproteins along the axis of mechanical load produced by the mechanicalforces or stimulation, if such forces are applied. In a preferredembodiment, the ligament or tendon is characterized by the presence offiber bundles which are arranged into a helical organization.

Another aspect of the present invention is a method for producing a widerange of ligament and tendon types ex vivo using the novelsilk-fiber-based matrix, and an adaptation of the method for producingan anterior cruciate ligament by adapting the matrix (e.g., geometry,organization, composition)(see Example 1) and anchor size to reflect thesize of the specific type of ligament or tendon to be produced (e.g.,posterior cruciate ligament, rotator cuff tendons, medial collateralligament of the elbow and knee, flexor tendons of the hand, lateralligaments of the ankle and tendons and ligaments of the jaw ortemporomandibular joint), and also adapting the specific combination offorces applied, to mimic the mechanical stimuli experienced in vivo bythe specific type of ligament or tendon to be produced. Similaradaptations of this method, and which are considered to be a part of theinvention, can be made to produce other tissues ex vivo from pluripotentstem cells, by adapting additional matrix compositions, geometries,organizations, and the mechanical forces applied during cell culture tomimic stresses experienced in vivo by the specific tissue type to beproduced. The methods of the present invention can be further modifiedto incorporate other stimuli experienced in vivo by the particulardeveloping tissue. Some examples of other stimuli include chemicalstimuli and electro-magnetic stimuli.

As used herein, the term “tissue” is intended to take on its generallyrecognized biological/medical definition to those of skill in the art.As a non-limiting example, “tissue” is defined in Stedman's MedicalDictionary as:

-   -   [A] collection of similar cells and the intercellular substances        surrounding them. There are four basic tissues in the body: 1)        epithelium; 2) the connective tissues, including blood, bone,        and cartilage; 3) muscle tissue; and 4) nerve tissue.

Another aspect of the present invention relates to the specificligaments or tendons which are produced by the methods of the presentinvention. Some examples of ligaments or tendons that can be producedinclude anterior cruciate ligament, posterior cruciate ligament, rotatorcuff tendons, medial collateral ligament of the elbow and knee, flexortendons of the hand, lateral ligaments of the ankle and tendons andligaments of the jaw or temporomandibular joint. Other tissues that maybe produced by methods of the present invention include cartilage (botharticular and meniscal), bone, muscle, skin and blood vessels.

The above description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be understood, and in order that the presentcontributions to the art may be better appreciated. Other objects andfeatures of the present invention will become apparent from thefollowing detailed description considered in conjunction with theaccompanying drawings. It is to be understood, however, that thedrawings are designed solely for the purposes of illustration and not asa definition of the limits of the invention, for which reference shouldbe made to the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a scanning electron microscopy (SEM) image of single nativesilk fiber having a sericin coating;

FIG. 1B illustrates SEM of the silk fiber of FIG. 1A extracted for 60min at 37° C.;

FIG. 1C illustrates SEM of the silk fiber of FIG. 1A extracted for 60min at 90° C. and illustrating complete removal of the sericin coating;

FIG. 1D is a chart showing ultimate tensile strength (UTS) as a functionof extraction conditions;

FIG. 2A illustrates a single cord of Matrix 1 having a wire-ropegeometry composed of two levels of twisting hierarchy. When six cordsare used in parallel (e.g., Matrix 1), the matrix has mechanicalproperties similar to a native ACL.

FIG. 2B illustrates a single cord of Matrix 2 having a wire-ropegeometry composed of three levels of twisting hierarchy. When six cordsare used in parallel (e.g., Matrix 2), the matrix has mechanicalproperties similar to a native ACL.

FIG. 3A illustrates load-elongation curves (N=5) for Matrix 1 formedfrom six parallel silk fibroin cords illustrated in FIG. 2A.

FIG. 3B is a chart of cycles to failure at UTS, 1680N, and 1200N loads(n=5 for each load) illustrating Matrix 1 fatigue data. Regressionanalysis of Matrix 1 fatigue data, when extrapolated to physiologicalload levels (400 N) to predicite number of cycles to failure in vivo,indicates a matrix life of 3.3 million cycles;

FIG. 3C illustrates load-elongation curves for Matrix 2 (N=3) formedfrom six parallel silk fibroin cords as illustrated in FIG. 2B;

FIG. 3D is a chart of cycles to failure at UTS, 2280N, 2100N and 1800Nloads (N=3 for each load) illustrating Matrix 2 fatigue data. Regressionanalysis of Matrix 2 fatigue data, when extrapolated to physiologicalload levels (400 N) to predicite number of cycles to failure in vivo,indicates a matrix life of greater than 10 million cycles;

FIG. 4A illustrates SEM of extracted silk fibroin prior to seeding withcells;

FIG. 4B illustrates SEM of bone marrow stromal cells seeded and attachedon silk fibroin immediately post seeding;

FIG. 4C illustrates SEM of bone marrow cells attached and spread on silkfibroin 1 day post seeding;

FIG. 4D illustrates SEM of bone marrow stromal cells seeded on silkfibroin 14 days post seeding forming an intact cell-extracellular matrixsheet;

FIG. 5A illustrates a 3 cm length of the silk fibroin cord illustratedin FIG. 2A and seeded with bone marrow stromal cells, cultured for 14days in a static environment and stained with MTT to show even cellcoverage of the matrix following the growth period;

FIG. 5B illustrates a control strand of silk fibroin cord 3 cm in lengthstained with MTT;

FIG. 6A is a chart illustrating bone marrow stromal cell proliferationon silk fibroin Matrix 1 determined by total cellular DNA over 21 dayculture period indicating a significant increase in cell proliferationafter 21 days of culture;

FIG. 6B is a bar graph illustrating bone marrow stromal cellproliferation on silk fibroin Matrix 2 determined by total cellular DNAover 14 day culture period indicating a significant increase in cellproliferation after 14 days of culture;

FIG. 7 illustrates the ultimate tensile strength of a 30 silk fiberextracted construct which is either seeded with bone marrow stromalcells or non-seeded over 21 days of culture in physiological growthconditions;

FIG. 8 illustrates gel eletrophoretic analysis of RT-PCR amplificationof selected markers over time. The gel shows upregulation in bothcollagen types I and III expression levels normalized to thehousekeeping gene, GAPDH by bone marrow stromal cell grown on Matrix 2over 14 days in culture. Collagen type II (as a marker for cartilage)and bone sialoprotein (as a marker of bone tissue formation) were notdetected indicating a ligament specific differentiation response by theBMSCs when cultured with Matrix 2.

FIG. 9 illustrates a single cord of Matrix 1 (not seeded at the time ofimplantation) following six weeks of implantation in vivo and used toreconstruct the medial collateral ligament (MCL) in a rabbit model. (A)shows Matrix 1 fibroin fibers surrounded by progenitor host cells andtissue ingrowth into the matrix and around the individual fibroin fibersvisualized by hematoxylin and eosin staining; (B) shows collagenoustissue ingrowth into the matrix and around the individual fibroin fibersvisualized by trichrome staining; and

FIG. 10 illustrates bone marrow stromal cells seeded and grown oncollagen fibers for (A) 1 day and (B) 21 days; (C) RT-PCR and gelelectrophoretic analysis of collagen I and III expression vs. thehousekeeping gene GAPDH: a=Collagen I, day 14; b=Collagen I, day 18;c=Collagen III, day 14; d=Collagen III, day 18; e=GAPDH, day 14;f=GAPDH, day 18. Collagen type II (as a marker for cartilage) and bonesialoprotein (as a marker of bone tissue formation) were not detectedindicating a ligament specific differentiation response; and

FIG. 11 illustrates real-time quantitative RT-PCR at 14 days whichyielded a transcript ratio of collagen I to collagen III, normalized toGAPDH, of 8.9:1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel silk-fiber-based matrixupon which pluripotent cells may be seeded and which proliferate anddifferentiate into ligament and tendon fibroblasts resulting in theformation of an anterior cruciate ligament (ACL), or other ligaments,tendons or tissues. The novel silk-fiber-based matrix is designed havingfibers in a wire-rope (twisted or braided-like) geometry, which exhibitsmechanical properties identical to a natural anterior cruciate ligament(see Table 1, infra) and where simple variations in matrix organizationand geometry can result in the formation of any desired ligament ortendon tissue (see Table 2, infra).

The present invention is also based on the finding that pluripotent bonemarrow stromal cells (BMSCs) isolated and cultured as described inExample 1, seeded on the silk-fiber-based matrix, and cultured in abioreactor under static conditions will undergo ligament and tendonspecific differentiation forming viable tissue. In addition, thehistomorphological properties of a bioengineered tissue produced invitro generated from pluripotent cells within a matrix are affected bythe direct application of mechanical force to the matrix during tissuegeneration. This discovery provides important new insights into therelationship between mechanical stress, biochemical and cellimmobilization methods and cell differentiation, and has applications inproducing a wide variety of ligaments, tendons and tissues in vitro frompluripotent cells.

One aspect of the present invention relates to a matrix comprised ofsilk fibers having a wire-rope (twisted or braided-like) geometry, asillustrated in FIGS. 2A and 2B.

As described in the Examples below, mechanical properties of the silkfibroin (as illustrated in FIG. 1A-C) were characterized and geometriesfor forming applicable matrices for ACL engineering were derived using atheoretical computational model (see FIG. 1D). A six-cord construct waschosen for use as an ACL replacement to increase matrix surface area andto enhance support for tissue in-growth. Two of several optimalconstruct geometrical hierarchies have been determined to comprise thefollowing: Matrix 1: 1 ACL prosthesis=6 parallel cords; 1 cord=3 twistedstrands (3 twists/cm); 1 strand=6 twisted bundles (3 twists/cm); 1bundle=30 parallel washed fibers; and Matrix 2: 1 ACL matrix=6 parallelcords; 1 cord=3 twisted strands (2 twists/cm); 1 strand=3 twistedbundles (2.5 twists/cm); 1 bundle=3 groups (3 twists/cm); 1 group=15parallel extracted silk fibroin fibers. The number of fibers andgeometries for Matrix 1 and Matrix 2 were selected such that the silkprostheses are similar to the ACL biomechanical properties in ultimatetensile strength, linear stiffness, yield point and % elongation atbreak, serving as a solid starting point for the development of a tissueengineered ACL. The ability to generate two matrices with differinggeometries both resulting in mechanical properties which mimicproperties of the ACL indicates that an infinite number of geometricalconfigurations exist to achieve the desired mechanical properties. Oneskilled in the art will recognize that an alternative geometry for anydesired ligament or tendon tissue may comprise any number, combinationor organization of cords, strands, bundles, groups and fibers (see Table2, infra) that result in a matrix construct with applicable mechanicalproperties that mimic those of the ligament or tendon desired. Forexample, one (1) ACL prosthesis may have any number of cords in parallelranging from a single cord to an infinite number of cords provided thereis a means of anchoring the final matrix in vitro or in vivo. Oneskilled in the art would also recognize that no limit exists to thenumber of twisting levels (where a single level is defined as either agroup, bundle, strand or cord) for a given geometry providing the matrixresults in the desired mechanical properties. Furthermore, one skilledin the art would realize the large degree of freedom in designing thematrix geometry and organization in engineering an ACL prosthesis, andwill therefore understand the utility of the developed theoreticalcomputational model to predict the matrix design of a desired ligamentor tendon tissue (see Example 1). One skilled in the art would,therefore, recognize that a variation in geometry (i.e., the number ofcords used to make a prosthesis or number of fibers in a group) could beused to generate matrices applicable to most ligaments and tendons. Forexample, for smaller ligaments or tendons of the hand, the geometry andorganization used to generate a single cord of Matrix 1(or two cords orthree cords, etc.) may be appropriate given the matrix's organizationresults in mechanical properties suitable for the particularphysiological environment.

The invention is not, however, limited with respect to the wire-ropegeometry as described, and any geometry or combinations of geometries(e.g., parallel, twisted, braided, mesh-like) can be used that resultsin matrix mechanical properties similar to the ACL (i.e., >2000 Nultimate tensile strength, between 100-600 N/mm linear stiffness for anative ACL or commonly used replacement graft such as the patellartendon with length between 26-30 mm) or to the desired ligament andtendon that is to be produced. The number of fibers and geometry of bothMatrix 1 and Matrix 2 were selected to generate mechanically appropriateACL matrices, or other desired ligament or tendon matrices (e.g.,posterior cruciate ligament (PCL)). For example, a single cord of thesix-cord Matrix 1 construct was used to reconstruct the medialcollateral ligament (MC) in a rabbit (see FIG. 9). The mechanicalproperties of the silk six-cord constructs of Matrix 1 and Matrix 2 aredescribed in Table 1 and in FIG. 3. Additional geometries and theirrelating mechanical properties are listed in Table 2 as an example ofthe large degree of design freedom that would result in a matrixapplicable in ACL tissue engineering in accordance with the presentinvention. TABLE 1 UTS Stiffness Yield Pt. Elongation (N) (N/mm) (N) (%)Silk 2337 +/− 72 354 +/− 26 1262 +/− 36 38.6 +/− 2.4 matrix 1 Silk 3407+/− 63 580 +/− 40  1647 +/− 214 29 +/− 4 Matrix 2 Human  2160 +/− 157⁴ 242 +/− 28⁴ ˜1200 ˜26-32% ACLMechanical properties for two different cords based on a cord length of3 cm.

TABLE 2 Twisting Level (# of twists/cm) Matrix 1 Matrix 2 Matrix 3Matrix 4 Matrix 5 Matrix 6 Matrix 7 # fibers per group 30 15 1300 180 2010 15 (0) (0) (0) (0) (0) (0) (0) # groups per bundle 6 3 3 3 6 6 3 (3)(3) (2) (3.5) (3) (3) (3) # bundles per strand 3 6 1 3 3 3 3 (3) (2.5)(0) (2) (2) (2.5) (2.5) # strands per cord 6 3 — 2 3 3 3 (0) (2.0) (0)(1) (2) (2) # cords per ACL — 6 — — 3 6 12 (0) (0) (0) (0) UTS (N) 23373407 2780 2300 2500 2300 3400 Stiffness (N/mm) 354 580 300 350 550 500550Examples of several geometry hierarchies that would result in suitablemechanical properties for replacement of the ACL.Note:Matrix 1 and 2 have been developed as shown in example 1; Matrix 3 wouldyield a single bundle prosthesis, Matrix 4 would yield a 2 strandprosthesis, Matrix 5 would yield a 3 cord prosthesis, Matrix 6 isanother variation of a 6 cord prosthesis, and Matrix 7 will yield a 12cord prosthesis.

Advantageously, the silk-fiber based matrix is comprised solely of silk.Types and sources of silk include the following: silks from silkworms,such as Bombyx mori and related species; silks from spiders, such asNephila clavipes; silks from genetically engineered bacteria, yeastmammalian cells, insect cells, and transgenic plants and animals; silksobtained from cultured cells from silkworms or spiders; native silks;cloned full or partial sequences of native silks; and silks obtainedfrom synthetic genes encoding silk or silk-like sequences. In their rawform, the native silk fibroin obtained from the Bombyx mori silkwormsare coated with a glue-like protein called sericin, which typically isextracted from the fibers before the fibers which make up the matrix areseeded with cells.

In an alternative embodiment, the matrix may be comprised of a compositeof: (1) silk and collagen fibers (2) silk and collagen foams, meshes, orsponges, or a composite of (3) silk fibroin fibers and silk foams,meshes, or sponges, (4) silk and biodegradable polymers (e.g.,cellulose, cotton, gelatin, poly lactide, poly glycolic,poly(lactide-co-glycolide), poly caproloactone, polyamides,polyanhydrides, polyaminoacids, polyortho esters, poly acetals,proteins, degradable polyurethanes, polysaccharides, polycyanoacrylates,Glycosamino glycans—e.g., chrondroitin sulfate, heparin, etc.,Polysaccharides—native, reprocessed or genetically engineeredversions—e.g. hyaluronic acid, alginates, xanthans, pectin, chitosan,chitin, and the like, Elastin—native, reprocessed or geneticallyengineered and chemical versions, Collagens—native, reprocessed orgenetically engineered versions), or (5) silk and non-biodegradablepolymers (e.g., polyamide, polyester, polystyrene, polypropylene,polyacrylate, polyvinyl, polycarbonate, polytetrafluorethylene, ornitrocellulose material. The composite generally enhances the matrixproperties such as porosity, degradability, and also enhances cellseeding, proliferation, differentiation or tissue development. FIG. 10illustrates the ability of collagen fibers to support BMSC growth andligament specific differentiation.

The matrix of the present invention may also be treated to enhance cellproliferation and/or tissue differentiation thereon. Exemplary matrixtreatments for enhancing cell proliferation and tissue differentiationinclude, but are not limited to, metals, irradiation, crosslinking,chemical surface modifications (e.g. RGD (arg-gly-asp) peptide coating,fibronectin coating, coupling growth factors), and physical surfacemodifications.

A second aspect of the present invention relates to a mechanically andbiologically functional ACL formed from a novel silk-fiber-based matrixand autologous or allogenic (depending on the recipient of the tissue)bone marrow stromal cells (BMSCs) seeded on the matrix. Thesilk-fiber-based matrix induces stromal cell differentiation towardsligament lineage without the need for any mechanical stimulation duringbioreactor cultivation. BMSCs seeded on the silk-fiber-based matrix andgrown in a petri dish begin to attach and spread (see FIG. 4),proliferate covering the matrix (see FIGS. 5 and 6) and differentiate asshown by the expression of ligament specific markers (see FIG. 8).Markers for cartilage (collagen type II) and for bone (bonesialoprotein) were not expressed (see FIG. 8). Data illustrating theexpression of ligament specific markers is set forth in Example 2.

In another aspect, the present invention relates to a method forproducing an ACL ex vivo. Cells capable of differentiating into ligamentcells are grown under conditions which simulate the movements and forcesexperienced by an ACL in vivo through the course of embryonicdevelopment into mature ligament function. This is accomplished by thefollowing steps: under sterile conditions, pluripotent cells are seededwithin a three dimensional silk-fiber-based matrix to which cells canadhere and which is advantageously of cylindrical shape. The threedimensional silk-fiber based matrix used in the method serves as apreliminary matrix, which is supplemented and possibly even replaced byextracellular matrix components produced by the differentiating cells.Use of the novel silk-fiber-based matrix may enhance or accelerate thedevelopment of the ACL. For instance, the novel silk-fiber-based matrixwhich has specific mechanical properties (e.g., increased tensilestrength) that can withstand strong forces prior to reinforcement fromcellular extracellular matrix components. Other advantageous propertiesof the novel silk-fiber based preliminary matrix include, withoutlimitation, biocompatibility and susceptibility to biodegradation.

The pluripotent cells may be seeded within the preliminary matrix eitherpre- or post-matrix formation, depending upon the particular matrix usedand the method of matrix formation. Uniform seeding is preferable. Intheory, the number of cells seeded does not limit the final ligamentproduced, however optimal seeding may increase the rate of generation.Optimal seeding amounts will depend on the specific culture conditions.In one embodiment, the matrix is seeded with from about 0.05 to 5 timesthe physiological cell density of a native ligament.

One or more types of pluripotent cells are used in the method. Suchcells have the ability to differentiate into a wide variety of celltypes in response to the proper differentiation signals and to expressligament specific markers. More specifically, the method requires theuse of cells that have the ability to differentiate into cells ofligament and tendon tissue. In a preferred embodiment, bone marrowstromal cells are used. If the resulting bioengineered ligament is to betransplanted into a patient, the cells should be derived from a sourcethat is compatible with the intended recipient. Although the recipientwill generally be a human, applications in veterinary medicine alsoexist. In one embodiment, the cells are obtained from the recipient(autologous), although compatible donor cells may also be used to makeallogenic ligaments. For example, when making allogenic ligaments (e.g.,using cells from another human such as bone marrow stromal cellsisolated from donated bone marrow or ACL fibroblasts isolated fromdonated ACL tissue), human anterior cruciate ligament fibroblast cellsisolated from intact donor ACL tissue ( e.g. cadaveric or from totalknee transplantations), ruptured ACL tissue (e.g., harvested at the timeof surgery from a patient undergoing ACL reconstruction) or bone marrowstromal cells may be used. The determination of compatibility is withinthe means of the skilled practitioner.

In alternative embodiments of the present invention, ligaments ortendons including, but not limited to, the posterior cruciate ligament,rotator cuff tendons, medial collateral ligament of the elbow and knee,flexor tendons of the hand, lateral ligaments of the ankle and tendonsand ligaments of the jaw or temporomandibular joint other than ACL,cartilage, bone and other tissues may be engineered with the matrix inaccordance with the method of the present invention. In this manner, thecells to be seeded on the matrix are selected in accordance with thetissue to be produced (e.g., pluripotent or of the desired tissue type).Cells seeded on the matrix in accordance with the present invention maybe autologous or allogenic. The use of autologous cells effectivelycreates an allograft or autograft for implantation in a recipient.

As recited, to form an ACL, cells, which are advantageously bone marrowstromal cells, are seeded on the matrix. Bone marrow stromal cells are atype of pluripotent cell, and are also referred to in the art asmesenchymal stem cells or simply as stromal cells. As recited, thesource of these cells can be autologous or allogenic. The presentinvention also contemplates the use of adult or embryonic stem orpluripotent cells, in so long as the proper environment (either in vivoor in vitro), seeded on the silk-fiber based matrix, can recapitulate anACL or any other desired ligament or tissue in extracellular matrixcomposition (e.g., protein, glycoprotein content) organization,structure or function.

Fibroblast cells are also contemplated by the present invention forseeding on the inventive matrix. Since fibroblast cells are often notreferred to as pluripotent cells, fibroblasts are intended to includemature human ACL fibroblasts (autologous or allogenic) isolated from ACLtissue, fibroblasts from other ligament tissue, fibroblasts from tendontissue, from netonatal foreskin, from umbilical cord blood, or from anycell, whether mature or pluripotent, mature dedifferentiated, orgenetically engineered, such that when cultured in the properenvironment (either in vivo or in vitro), and seeded on the silk-fiberbased matrix, can recapitulate an ACL or any other desired ligament ortissue in extracellular matrix composition (e.g., protein, glycoproteincontent), organization, structure or function.

The faces of the matrix cylinder are each attached to anchors, throughwhich a range of forces are to be applied to the matrix. To facilitateforce delivery to the matrix, it is preferable that the entire surfaceof each respective face of the matrix contact the face of the respectiveanchors. Anchors with a shape which reflects the site of attachment(e.g., cylindrical) are best suited for use in this method. Onceassembled, the cells in the anchored matrix are cultured underconditions appropriate for cell growth and regeneration. The matrix issubjected to one or more mechanical forces applied through the attachedanchors (e.g., via movement of one or both of the attached anchors)during the course of culture. The mechanical forces are applied over theperiod of culture to mimic conditions experienced by the native ACL, orother tissues in vivo.

The anchors must be made of a material suitable for matrix attachment,and the resulting attachment should be strong enough to endure thestress of the mechanical forces applied. In addition, it is preferablethat the anchors be of a material which is suitable for the attachmentof extracellular matrix which is produced by the differentiating cells.The anchors support bony tissue in-growth (either in vitro or in vivo)while anchoring the developing ligament. Some examples of suitableanchor material include, without limitation, hydroxyappatite, Goinopracoral, demineralized bone, bone (allogenic or autologous). Anchormaterials may also include titanium, stainless steel, high densitypolyethylene, Dacron and Teflon.

Alternatively, anchor material may be created or further enhanced byinfusing a selected material with a factor which promotes eitherligament matrix binding or bone matrix binding or both. The term infuseis considered to include any method of application which appropriatelydistributes the factor onto the anchor (e.g., coating, permeating,contacting). Examples of such factors include without limitation,laminin, fibronectin, any extracellular matrix protein that promotesadhesion, silk, factors which contain arginine-glycine-aspartate (RGD)peptide binding regions or the RGD peptides themselves. Growth factorsor bone morphogenic protein can also be used to enhance anchorattachment. In addition, anchors may be pre-seeded with cells (e.g.,stem cells, ligament cells, osteoblasts, osteogenic progenitor cells)which adhere to the anchors and bind the matrix, to produce enhancedmatrix attachment both in vitro and in vivo.

An exemplary anchor system is disclosed in applicant's co-pendingapplication U.S. Ser. No. 09/______. The matrix is attached to theanchors via contact with the anchor face or alternatively by actualpenetration of the matrix material through the anchor material. Becausethe force applied to the matrix via the anchors dictates the finalligament produced, the size of the final ligament produced is, in part,dictated by the size of the attachment site of the anchor. One of skillin the art will appreciate that an anchor of appropriate size to thedesired final ligament should be used. A preferred anchor shape for theformation of an ACL is a cylinder. However, one of skill in the art willappreciate that other anchor shapes and sizes will also functionadequately. In a preferred embodiment, anchors have an appropriate sizeand composition for direct insertion into bone tunnels in the femur andtibia of a recipient of the bioengineered ligament.

In an alternative embodiment of the present invention, anchors may beused only temporarily during in vitro culture, and then removed when thematrix alone is implanted in vivo.

In another embodiment, the novel silk-fiber-based matrix is seeded withBMSCs and cultured in a bioreactor. Two types of growth environmentscurrently exist that may be used in accordance with this invention: (1)the in vitro bioreactor apparatus system, and (2) the in vivo kneejoint, which serves as a “bioreactor” as it provides the physiologicenvironment including progenitor cells and stimuli (both chemical andphysical) necessary for the development of a viable ACL given a matrixwith proper biocompatible and mechanical properties. The bioreactorapparatus provides optimal culture conditions for the formation of aligament in terms of differentiation and extracellular matrix (ECM)production, and which thus provides the ligament with optimal mechanicaland biological properties prior to implantation in a recipient.Additionally, when the silk-fiber based matrix is seeded and culturedwith cells in vitro, a petri dish may be considered to be the bioreactorwithin which conditions appropriate for cell growth and regenerationexist, i.e., a static environment.

In accordance with the one embodiment of present invention, cells mayalso be cultured on the matrix without the application of any mechanicalforces, i.e., in a static environment. For example, the silk-fiber basedmatrix alone, with no in vitro applied mechanical forces or stimulation,when seeded and cultured with BMSCs, induces the cells to proliferateand express ligament and tendon specific markers (See Example 2 and FIG.8). The knee joint may serve as a physiological growth and developmentenvironment that can provide the cells and the correct environmentalsignals (chemical and physical) to the matrix such that an ACLtechnically develops. Therefore, the knee joint (as its own form ofbioreactor) plus the matrix (either non-seeded, seeded and notdifferentiated in vitro, or seeded and differentiated in vitro prior toimplantation) will result in the development of an ACL, or other desiredtissue depending upon the cell type seeded on the matrix and theanatomical location of matrix implantation. FIG. 9 illustrates theeffects of the medial collateral knee joint environment on medialcollateral ligament (MCL) development when only a non-seeded silk-basedmatrix with appropriate MCL mechanical properties is implanted for 6weeks in vivo. Whether the cells are cultured in a static environmentwith no mechanical stimulation applied, or in a dynamic environment,such as in a bioreactor apparatus, conditions appropriate for cellgrowth and regeneration are advantageously present for the engineeringof the desired ligament or tissue.

In the experiments described in the Examples section below, the appliedmechanical stimulation was shown to influence the morphology, andcellular organization of the progenitor cells within the resultingtissue. The extracellular matrix components secreted by the cells andorganization of the extracellular matrix throughout the tissue was alsosignificantly influenced by the forces applied to the matrix duringtissue generation. During in vitro tissue generation, the cells andextracellular matrix aligned along the axis of load, reflecting the invivo organization of a native ACL which is also along the various loadaxes produced from natural knee joint movement and function. Theseresults suggest that the physical stimuli experienced in nature by cellsof developing tissue, such as the ACL, play a significant role inprogenitor cell differentiation and tissue formation. They furtherindicate that this role can be effectively duplicated in vitro bymechanical manipulation to produce a similar tissue. The more closelythe forces produced by mechanical manipulation resemble the forcesexperienced by an ACL in vivo, the more closely the resultant tissuewill resemble a native ACL.

When mechanical stimulation is applied in vitro to the matrix via abioreactor, there exists independent but concurrent control over bothcyclic and rotation strains as applied to one anchor with respect to theother anchor. In an alternative embodiment, the matrix alone may beimplanted in vivo, seeded with ACL cells from the patient and exposed invivo to mechanical signaling via the patient.

When the matrix is seeded with cells prior to implantation, the cellsare cultured within the matrix under conditions appropriate for cellgrowth and differentiation. During the culture process, the matrix maybe subjected to one or more mechanical forces via movement of one orboth of the attached anchors. The mechanical forces of tension,compression, torsion and shear, and combinations thereof, are applied inthe appropriate combinations, magnitudes, and frequencies to mimic themechanical stimuli experienced by an ACL in vivo.

Various factors will influence the amount of force which can betolerated by the matrix (e.g., matrix composition, cell density). Matrixstrength is expected to change through the course of tissue development.Therefore, mechanical forces or strains applied will increase, decreaseor remain constant in magnitude, duration, frequency and variety overthe period of ligament generation, to appropriately correspond to matrixstrength at the time of application.

When producing an ACL, the more accurate the intensity and combinationof stimuli applied to the matrix during tissue development, the more theresulting ligament will resemble a native ACL. Two issues must beconsidered regarding the natural function of the ACL when devising thein vitro mechanical force regimen that closely mimics the in vivoenvironment: (1) the different types of motion experienced by the ACLand the responses of the ACL to knee joint movements and (2) the extentof the mechanical stresses experienced by the ligament. Specificcombinations of mechanical stimuli are generated from the naturalmotions of the knee structure and transmitted to the native ACL.

To briefly describe the motions of the knee, the connection of the tibiaand femur by the ACL between provides six degrees of freedom whenconsidering the motions of the two bones relative to each other: thetibia can move in the three directions and can rotate relative to theaxes for each of these three directions. The knee is restricted fromachieving the full ranges of these six degrees of freedom due to thepresence of ligaments and capular fibers and the knee surfacesthemselves (Biden et al., Experimental methods used to evaluate kneeligament function. In Knee Ligaments: Structure, Function. Injury andRepair, Ed. D. Daniel et al. Raven Press, pp. 135-151 (1990)). Smalltranslational movements are also possible. The attachment sites of theACL are responsible for its stabilizing roles in the knee joint. The ACLfunctions as a primary stabilizer of anterior-tibial translation, and asa secondary stabilizer of valgus-varus angulation, and tibial rotation(Shoemaker et al., The limits of knee motion. In Knee Ligaments:Structure, Function, Injury and Repair, Ed. D. Daniel et al. RavenPress, pp. 1534-161 (1990)). Therefore, the ACL is responsible forstabilizing the knee in three of the six possible degrees of freedom. Asa result, the ACL has developed a specific fiber organization andoverall structure to perform these stabilizing functions. The presentinvention simulates these conditions in vitro to produce a tissue withsimilar structure and fiber organization.

The extent of mechanical stresses experienced by the ACL can besimilarly summarized. The ACL undergoes cyclic loads of about 400 Nbetween one and two million cycles per year (Chen et al., J. Biomed.Mat. Res. 14: 567-586 (1980). It is also critical to consider linearstiffness (˜182 N/mm), ultimate deformation (100% of ACL) and energyabsorbed at failure (12.8 N-m) (Woo et al., The tensile properties ofhuman anterior cruciate ligament (ACL) and ACL graft tissues. In KneeLigaments: Structure, Function, Injury and Repair, Ed. D. Daniel et al.Raven Press, pp. 279-289 (1990)) when developing an ACL surgicalreplacement.

The Examples section below details the production of a prototypebioengineered anterior cruciate ligament (ACL) ex vivo. Mechanicalforces mimicking a subset of the mechanical stimuli experienced by anative ACL in vivo (rotational deformation and linear deformation) wereapplied in combination, and the resulting ligament which was formed wasstudied to determine the effects of the applied forces on tissuedevelopment. Exposure of the developing ligament to physiologicalloading during in vitro formation induced the cells to adopt a definedorientation along the axes of load, and to generate extracellularmatrices along the axes as well. These results indicate that theincorporation of complex multi-dimensional mechanical forces into theregime to produce a more complex network of load axes that mimics theenvironment of the native ACL, will produce a bioengineered ligamentwhich more closely resembles a native ACL.

The different mechanical forces that may be applied include, withoutlimitation, tension, compression, torsion, and shear. These forces areapplied in combinations which simulate forces experienced by an ACL inthe course of natural knee joint movements and function. These movementsinclude, without limitation, knee joint extension and flexion as definedin the coronal and sagittal planes, and knee joint flexion. Optimally,the combination of forces applied mimics the mechanical stimuliexperienced by an anterior cruciate ligament in vivo as accurately as isexperimentally possible. Varying the specific regimen of forceapplication through the course of ligament generation is expected toinfluence the rate and outcome of tissue development, with optimalconditions to be determined empirically. Potential variables in theregimen include, without limitation: (1) strain rate, (2) percentstrain, (3) type of strain, e.g. translation and rotation, (4)frequency, (5) number of cycles within a given regime, (6) number ofdifferent regimes, (7) duration at extreme points of ligamentdeformation, (8) force levels, and (9) different force combinations. Itwill be recognized by one of skill in the art that a potentiallyunlimited number of variations exist. In a preferred embodiment theregimen of mechanical forces applied produces helically organized fiberssimilar to those of the native ligament, described below.

The fiber bundles of a native ligament are arranged into a helicalorganization. The mode of attachment and the need for the knee joint torotate ˜140° of flexion has resulted in the native ACL inheriting a 90°twist and with the peripheral fiber bundles developing a helicalorganization. This unique biomechanical feature allows the ACL tosustain extremely high loading. In the functional ACL, this helicalorganization of fibers allows anterior-posterior and posterior-anteriorfibers to remain relatively isometric in respect to one another for alldegrees of flexion, thus load can be equally distributed to all fiberbundles at any degree of knee joint flexion stabilizing the kneethroughout all ranges of joint motion. In a preferred embodiment of theinvention, mechanical forces that simulate a combination of knee jointflexion and knee joint extension are applied to the developing ligamentto produce an engineered ACL which possesses this same helicalorganization. The mechanical apparatus used in the experiments presentedin the Examples below provides control over strain and strain rates(both translational and rotational). The mechanical apparatus willmonitor the actual load experienced by the growing ligaments, serving to‘teach’ the ligaments over time through monitoring and increasing theloading regimes.

Another aspect of the present invention relates to the bioengineeredanterior cruciate ligament produced by the above described methods. Thebioengineered ligament produced by these methods is characterized bycellular orientation and/or a matrix crimp pattern in the direction ofthe mechanical forces applied during generation. The ligament is alsocharacterized by the production/presence of extracellular matrixcomponents (e.g., collagen type I, and type III, fibronectin, andtenascin-C proteins) along the axis of mechanical load experiencedduring culture. In a preferred embodiment, the ligament fiber bundlesare arranged into a helical organization, as discussed above.

The above methods using the novel silk-fiber-based matrix are notlimited to the production of an ACL, but can also be used to produceother ligaments and tendons found in the knee (e.g., posterior cruciateligament) or other parts of the body (e.g., hand, wrist, ankle, elbow,jaw and shoulder), such as for example, but not limited to posteriorcruciate ligament, rotator cuff tendons, medial collateral ligament ofthe elbow and knee, flexor tendons of the hand, lateral ligaments of theankle and tendons and ligaments of the jaw or temporomandibular joint.All moveable joints in a human body have specialized ligaments whichconnect the articular extremities of the bones in the joint. Eachligament in the body has a specific structure and organization which isdictated by its function and environment. The various ligaments of thebody, their locations and functions are listed in Anatomy, Descriptiveand Surgical (Gray, H., Eds. Pick, T. P., Howden, R., Bounty Books, NewYork (1977)), the pertinent contents of which are incorporated herein byreference. By determining the physical stimuli experienced by a givenligament or tendon, and incorporating forces which mimic these stimuli,the above described method for producing an ACL ex vivo can be adaptedto produce bioengineered ligaments and tendons ex vivo which simulatesany ligament or tendon in the body.

The specific type of ligament or tendon to be produced is predeterminedprior to tissue generation since several aspects of the method vary withthe specific conditions experienced in vivo by the native ligament ortendon. The mechanical forces to which the developing ligament or tendonis subjected during cell culture are determined for the particularligament or tendon type being cultivated. The specific conditions can bedetermined by those skilled in the art by studying the native ligamentor tendon and its environment and function. One or more mechanicalforces experienced by the ligament or tendon in vivo are applied to thematrix during culture of the cells in the matrix. The skilledpractitioner will recognize that a ligament or tendon which is superiorto those currently available can be produced by the application of asubset of forces experienced by the native ligament or tendon. However,optimally, the full range of in vivo forces will be applied to thematrix in the appropriate magnitudes and combinations to produce a finalproduct which most closely resembles the native ligament or tendon.These forces include, without limitation, the forces described above forthe production of an ACL. Because the mechanical forces applied varywith ligament or tendon type, and the final size of the ligament ortendon will be influenced by the anchors used, optimal anchorcomposition, size and matrix attachment sites are to be determined foreach type of ligament or tendon by the skilled practitioner. The type ofcells seeded on the matrix is obviously determined based on the type ofligament or tendon to be produced.

Another aspect of the present invention relates to the production ofother tissue types ex vivo using methods similar to those describedabove for the generation of ligaments or tendons ex vivo. The abovedescribed methods can also be applied to produce a range of engineeredtissue products which involve mechanical deformation as a major part oftheir function, such as muscle (e.g., smooth muscle, skeletal muscle,cardiac muscle), bone, cartilage, vertebral discs, and some types ofblood vessels. Bone marrow stomal cells possess the ability todifferentiate into these as well as other tissues. The geometry of thesilk-based matrix or composite matrix can easily be adapted to thecorrect anatomical geometrical configuration of the desired tissue type.For example, silk fibroin fibers can be reformed in a cylindrical tubeto recreate arteries.

The results presented in the Examples below indicate that growth in anenvironment which mimics the specific mechanical environment of a giventissue type will induce the appropriate cell differentiation to producea bioengineered tissue which significantly resembles native tissue. Theranges and types of mechanical deformation of the matrix can be extendedto produce a wide range of tissue structural organization. Preferably,the cell culture environment reflects the in vivo environmentexperienced by the native tissue and the cells it contains, throughoutthe course of embryonic development to mature function of the cellswithin the native tissue, as accurately as possible. Factors to considerwhen designing specific culture conditions to produce a given tissueinclude, without limitation, the matrix composition, the method of cellimmobilization, the anchoring method of the matrix or tissue, thespecific forces applied, and the cell culture medium. The specificregimen of mechanical stimulation depends upon the tissue type to beproduced, and is established by varying the application of mechanicalforces (e.g., tension only, torsion only, combination of tension andtorsion, with and without shear, etc.), the force amplitude (e.g., angleor elongation), the frequency and duration of the application, and theduration of the periods of stimulation and rest.

The method for producing the specific tissue type ex vivo is anadaptation of the above described method for producing an ACL.Components involved include pluripotent cells, a three-dimensionalmatrix to which cells can adhere, and a plurality of anchors which havea face suitable for matrix attachment. The pluripotent cells (preferablybone marrow stromal cells) are seeded in the three dimensional matrix bymeans to uniformly immobilize the cells within the matrix. The number ofcells seeded is also not viewed as limiting, however, seeding the matrixwith a high density of cells may accelerate tissue generation.

The specific forces applied are to be determined for each tissue typeproduced through examination of native tissue and the mechanical stimuliexperienced in vivo. A given tissue type experiences characteristicforces which are dictated by location and function of the tissue withinthe body. For instance, cartilage is known to experience a combinationof shear and compression/tension in vivo, bone experiences compression.Determination of the specific mechanical stimuli experienced in vivo bya given tissue is within the means of one of skill in the art.

Additional stimuli (e.g., chemical stimuli, electro-magnetic stimuli)can also be incorporated into the above described methods for producingbioengineered ligaments, tendons and other tissues. Cell differentiationis known to be influenced by chemical stimuli from the environment,often produced by surrounding cells, such as secreted growth ordifferentiation factors, cell-cell contact, chemical gradients, andspecific pH levels, to name a few. Other more unique stimuli areexperienced by more specialized types of tissues (e.g., the electricalstimulation of cardiac muscle). The application of such tissue specificstimuli (e.g., 1-10 ng/ml transforming growth factor beta-1 (TGF-β1)independently or in concert with the appropriate mechanical forces isexpected to facilitate differentiation of the cells into a tissue whichmore closely approximates the specific natural tissue.

Tissues produced by the above described methods provide an unlimitedpool of tissue equivalents for surgical implantation into a compatiblerecipient, particularly for replacement or repair of damaged tissue.Engineered tissues may also be utilized for in vitro studies of normalor pathological tissue function, e.g., for in vitro testing of cell- andtissue-level responses to molecular, mechanical, or geneticmanipulations. For example, tissues based on normal or transfected cellscan be used to assess tissue responses to biochemical or mechanicalstimuli, identify the functions of specific genes or gene products thatcan be either over-expressed or knocked-out, or to study the effects ofpharmacological agents. Such studies will likely provide more insightinto ligament, tendon and tissue development, normal and pathologicalfunction, and eventually lead toward fully functional tissue engineeredreplacements, based in part on already established tissue engineeringapproaches, new insights into cell differentiation and tissuedevelopment, and the use of mechanical regulatory signals in conjunctionwith cell-derived and exogenous biochemical factors to improvestructural and functional tissue properties.

The production of engineered tissues such as ligaments and tendons alsohas the potential for applications such as harvesting bone marrow stomalcells from individuals at high risk for tissue injury (e.g., ACLrupture) prior to injury. These cells could be either stored untilneeded or seeded into the appropriate matrix and cultured anddifferentiated in vitro under mechanical stimuli to produce a variety ofbioengineered prosthetic tissues to be held in reserve until needed bythe donor. The use of bioengineered living tissue prosthetics thatbetter match the biological environment in vivo, provide the requiredphysiological loading to sustain for example, the dynamic equilibrium ofa normal, fully functional ligament, should reduce rehabilitation timefor a recipient of a prosthesis from months to weeks, particularly ifthe tissue is pre-grown and stored. Benefits include a more rapid regainof functional activity, shorter hospital stays, and fewer problems withtissue rejections and failures.

It is to be understood that the present invention is not intended to belimited to a silk-fiber-based matrix for producing ACL, and otherligaments and tendons, as well as other tissues, such as cartilage,bone, skin and blood vessels are contemplated by the present inventionby utilizing the novel silk-fiber based matrix seeded with theappropriate cells and exposed to the appropriate mechanical stimulationif necessary, for proliferating and differentiating into the desiredligament, tendon or tissue.

Additionally, the present invention is not limited to using bone marrowstromal cells for seeding on the matrix, and other progenitor,pluripotent and stem cells, such as those in bone, muscle and skin forexample, may also be used to differentiate into ligaments and othertissues.

The invention is further defined by reference to the following examples.It will be apparent to those skilled in the art that many modifications,both to the materials and methods, may be practiced without departingfrom the purpose and interest of the invention.

EXAMPLES Example 1

a. Preparation of Silk Films

Raw Bombyx mori silkworm fibers, shown in FIG. 1A, were extracted toremove sericin, the glue-like protein coating the native silk fibroin(see FIG. 1A-C). The appropriate number of fibers per group werearranged in parallel and extracted in an aqueous solution of 0.02MNa₂CO₃ and 0.3% (w/v) Ivory soap solution for 60 min at 90° C., thenrinsed thoroughly with water to extract the glue-like sericin proteins.

b. Preparation of and Properties of the Silk-Fiber Based MatrixConstruct

Costello's equation for a three-strand wire rope was derived to predictmechanical properties of the silk-fiber-based matrix. The derived modelis a series of equations that when combined, take into account extractedsilk fiber material properties and desired matrix geometrical hierarchyto compute the overall strength and stiffness of the matrix as afunction of pitch angle for a given level of geometrical hierarchy

The material properties of a single silk fiber include fiber diameter,modulus of elasticity, Poisson's ratio, and the ultimate tensilestrength (UTS). Geometrical hierarchy may be defined as the number oftwisting levels in a given matrix level. Each level (e.g., group,bundle, strand, cord, ligament) is further defined by the number ofgroups of fibers twisted about each other and the number of fibers ineach group of the first level twisted where the first level is define asa group, the second level as a bundle, the third as a strand and thefourth as a cord, the fifth as the ligament).

The model assumes that each group of multiple fibers act as a singlefiber with an effective radius determined by the number of individualfibers and their inherent radius, i.e., the model discounts frictionbetween the individual fibers due to its limited role in given arelatively high pitch angle.

Two applicable geometries (Matrix 1 and Matrix 2) of the many matrixgeometrical configurations (see Table 2, supra) computed to yieldmechanical properties mimicking those of a native ACL were derived formore detailed analysis. A six-cord construct was selected for use as theACL replacement. Matrix configurations are as follows: Matrix 1: 1 ACLprosthesis=6 parallel cords; 1 cord=3 twisted strands (3 twists/cm); 1strand=6 twisted bundles (3 twists/cm); 1 bundle=30 parallel washedfibers; and Matrix 2: 1 ACL matrix=6 parallel cords; 1 cord=3 twistedstrands (2 twists/cm); 1 strand=3 twisted bundles (2.5 twists/cm); 1bundle=3 groups (3 twists/cm); 1 group=15 parallel extracted silkfibroin fibers. The number of fibers and geometries were selected suchthat the silk prostheses are similar to the ACL biomechanical propertiesin UTS, linear stiffness, yield point and % elongation at break (seeTable 2, supra), thus serving as a solid starting point for thedevelopment of a tissue engineered ACL.

Mechanical properties of the silk fibroin were characterized using aservohydralic Instron 8511 tension/compression system with Fast-Tracksoftware (see FIG. 1D). Single pull-to-failure and fatigue analysis wereperformed on single silk fibers, extracted fibroin and organized cords.Fibers and fibroin were organized in both parallel and wire-ropegeometries (Matrix 1 (see FIG. 2A) and Matrix 2 (see FIG. 2B)) forcharacterization. Single pull to failure testing was performed at astrain rate of 100%/sec; force elongation histograms were generated anddata analyzed using Instron Series IX software. Both Matrix 1 and Matrix2 yielded similar mechanical and fatigue properties to the ACL in UTS,linear stiffness, yield point and % elongation at break (see Table 2 andFIG. 3).

Fatigue analyses were performed using a servohydraulic Instron 8511tension/compression system with Wavemaker software on single cords ofboth Matrix 1 and Matrix 2. Data was extrapolated to represent the6-cord ACL prostheses, which is shown in FIGS. 3B and 3D. Cord ends wereembedded in an epoxy mold to generate a 3 cm long construct betweenanchors. Cycles to failure at UTS, 1,680N and 1,200N (N=5 for each load)for Matrix 1 (see FIG. 3B) and at UTS, 2280N, 2100N and 1800N loads (n=3for each load) for Matrix 2 (see FIG. 3D) were determined using a H-sinewave function at 1 Hz generated by Wavemaker 32 version 6.6 (Instron,Canton, Mass.). Fatigue testing was conducted in a neutral phosphatebuffered saline (PBS) solution at room temperature.

Results

Complete sericin removal was observed after 60 min at 90° C. asdetermined by SEM (see FIGS. 1A-C). Removal of sericin from silk fibersaltered the ultrastructure of the fibers, resulting in a smoother fibersurface and the underlying silk fibroin was revealed (shown in FIGS.1A-C), with average diameter ranging between 20-40 μm. The fibroinexhibited a significant 15.2% decrease in ultimate tensile strength(1.033+/−0.042 N/fiber to 0.876+/−0.1 N/fiber) (p<0.05, paired Studentst-test) (see FIG. 1D). The mechanical properties of the optimized silkmatrices (see FIG. 2) are summarized in Table 1 above and in FIG. 3A(for Matrix 1) and in FIG. 3C (for Matrix 2). It is evident from theseresults that the optimized silk matrices exhibited values comparable tothose of native ACL, which have been reported to have an averageultimate tensile strength (UTS) of ˜2100 N, stiffness of ˜250 N/nm,yield point ˜2100 N and 33% elongation at break (See Woo, S L-Y, et al.,The Tensile Properties of Human Anterior Cruciate Ligament (ACL) and ACLGraft Tissue in Knee Ligaments: Structure, Function, Injury and Repair,279-289, Ed. D. Daniel et al., Raven Press 1990).

Regression analysis of matrix fatigue data, shown in FIG. 3B for Matrix1 and in FIG. 3D for Matrix 2, when extrapolated to physiological loadlevels (400 N) predict the number of cycles to failure in vivo, indicatea matrix life of 3.3 million cycles for Matrix 1 and a life of >10million cycles for Matrix 2. The wire rope matrix design utilizingwashed silk fibers resulted in a matrix with physiologically equivalentstructural properties, confirming its suitability as a scaffold forligament tissue engineering

Example 2 Cell Isolation and Culture

Bone Marrow Stromal Cells (BMSC), pluripotent cells capable ofdifferentiating into osteogenic, chondrogenic, tendonogenic, adipogenicand myogenic lineages, were chosen since the formation of theappropriate conditions can direct their differentiation into the desiredligament fibroblast cell line (Markolf et al., J. Bone Joint Surg. 71A:887-893 (1989); Caplan et al., Mesenchymal stem cells and tissue repair.In The Anterior Cruciate Ligament: Current and Future Concepts, Ed. D.W. Jackson et al., Raven Press, Ltd, New York (1993); Young et al., J.Orthopaedic Res. 16: 406-413 (1998)).

Human BMSCs were isolated from bone marrow from the iliac crest ofconsenting donors ≦25 years of age by a commercial vendor (Cambrex,Walkersville, Md.). Twenty-two milliliters of human marrow wasaseptically aspirated into a 25 ml syringe containing three millilitersof heparinized (1000 units per milliliter) saline solution. Theheparinized marrow solution was shipped overnight on ice to thelaboratory for bone marrow stromal cells isolation and culture. Uponarrival from the vendor, the twenty-five milliliter aspirates wereresuspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 100 U/mlpenicillin, 100 mg/L streptomycin (P/S), and 1 ng/ml basic fibroblastgrowth factor (bFGF) (Life Technologies, Rockville, Md.) and plated at8-10 microliters of aspirate/cm² in tissue culture flasks. Fresh mediumwas added to the marrow apirates twice a week for up to nine days ofculture. BMSCs were selected based on their ability to adhere to thetissue culture plastic; non-adherent hematopoietic cells were removedduring medium replacement after 9-12 days in culture. Medium was changedtwice per week thereafter. When primary BMSC became near confluent(12-14 days), they were detached using 0.25% trypsin/1 mM EDTA andreplated at 5×10³ cells/cm². First passage (P1) hBMSCs were trypsinizedand frozen in 8% DMSO/10% FBS/DMEM for future use.

Silk Matrix Cell Seeding

Frozen P1 hBMSCs were defrosted, replated at 5×10³ cells/cm² (P2),trypsinized when near confluency, and used for matrix seeding.Sterilized (ethylene oxide) silk matrices (specifically single cords ofMatrix 1 & 2, bundles of 30 parallel extracted silk fibers andwire-ropes of collage fibers) were seeded with cells in customizedseeding chambers (1 ml total volume) machined in Teflon blocks tominimize cell-medium volume and increase cell-matrix contact. Seededmatrices, following a 4 hour incubation period with the cell slurry(3.3×10⁶ BMSCs/ml) were transferred into a petri dish contain anappropriate amount of cell culture medium for the duration of theexperiments.

To determine the degradation rate of the silk fibroin, ultimate tensilestrength (UTS) was measured as a function of cultivation period inphysiological growth conditions, i.e., in cell culture medium. Groups of30 parallel silk fibers 3 cm in length were extracted, seeded withhBMSCs, and cultured on the fibroin over 21 days at 37° C. and 5% CO₂.Non-seeded control groups were cultured in parallel. Silk fibroin UTSwas determined as a function of culture duration for seeded andnon-seeded groups.

Results

The response of bone marrow stromal cells to the silk matrix was alsoexamined.

BMSCs readily attached and grew on the silk and collagen matrices after1 day in culture (See FIG. 4A-C and FIG. 10A), and formed cellularextensions to bridge neighboring fibers. As shown in FIG. 4D and FIG.10B, a uniform cells sheet covering the construct was observed at 14 and21 days of culture, respectively. MTT analysis confirmed complete matrixcoverage by seeded BMSCs after 14 days in culture (see FIG. 5). TotalDNA quantification of cells grown on Matrix 1 (see FIG. 6A) and Matrix 2(see FIG. 6B) confirmed that BMSCs proliferated and grew on the silkconstruct with the highest amount of DNA measured after 21 and 14 days,respectively, in culture.

Both BMSC seeded or non-seeded extracted control silk fibroin groups of30 fibers, maintained their mechanical integrity as a function ofculture period over 21 days (see FIG. 7).

RT-PCR analysis of BMSCs seeded on cords of Matrix 2 indicated that bothcollagen I & III were upregulated over 14 days in culture (FIG. 8).Collagen type II and bone sialoprotein (as indicators of cartilage andbone specific differentiation, respectively) were either not detectableor negligibly expressed over the cultivation period. Real-timequantitative RT-PCR at 14 days yielded a transcript ratio of collagen Ito collagen III, normalized to GAPDH, of 8.9:1 (see FIG. 11). The highratio of collagen I to collagen III indicates that the response is notwound healing or scar tissue formation (as is observed with high levelsof collagen type III), but rather ligament specific; the relative ratioof collagen I to collagen III in a native ACL is ˜6.6:1 (Amiel et al.,In Knee Ligaments: Structure, Function, Injury, and Repair. 1990).

Example 3

Studies are conducted to provide insight into the influence of directedmulti-dimensional mechanical stimulation on ligament formation from bonemarrow stromal cells in the bioreactor system. The bioreactor is capableof applying independent but concurrent cyclic multi-dimensional strains(e.g., translation, rotation) to the developing ligaments. After a 7 to14 day static rest period (time post seeding), the rotational andtranslation strain rates and linear and rotational deformation are keptconstant for 1 to 4 weeks. Translational strain (3.3%-10%, 1-3 mm) androtational strain (25%, 90°) are concurrently applied at a frequency of0.0167 Hz (one full cycle of stress and relaxation per minute) to thesilk-based matrices seeded with BMSCs; an otherwise identical set ofbioreactors with seeded matrices without mechanical loading serve ascontrols. The ligaments are exposed to the constant cyclic strains forthe duration of the experiment days.

Following the culture period, ligament samples, both the mechanicallychallenged as well as the controls (static) are characterized for: (1)general histomorphological appearance (by visual inspection); (2) celldistribution (image processing of histological and MTT stainedsections); (3) cell morphology and orientation (histological analysis);and (4) the production of tissue specific markers (RT-PCR,immunostaining).

Mechanical stimulation markedly affects the morphology and organizationof the BMSCs and newly developed extracellular matrix, the distributionof cells along the matrix, and the upregulation of a ligament-specificdifferentiation cascade; BMSCs align along the long axis of the fiber,take on a spheroid morphology similar to ligament/tendon fibroblasts andupregulate ligament/tendon specific markers. Newly formed extracellularmatrix is expected to align along the lines of load as well as the longaxis of the matrix. Directed mechanical stimulation is expected toenhance ligament development and formation in vitro in a bioreactorresulting from BMSCs seeded on the novel silk-based matrix. Thelongitudinal orientation of cells and newly formed matrix is similar toligament fibroblasts found within an ACL in vivo (Woods et al. Amer. J.Sports Med. 19: 48-55 (1991)). Furthermore, mechanical stimulationmaintains the correct expression ratio between collagen I transcriptsand collagen type III transcripts (e.g., >7:1) indicating the presenceof newly formed ligament tissue versus scare tissue formation. The aboveresults will indicate that the mechanical apparatus and bioreactorsystem provide a suitable environment (e.g., multi-dimensional strains)for in vitro formation of tissue engineered ligaments starting from bonemarrow stromal cells and the novel silk-based matrix.

The culture conditions used in these preliminary experiments can befurther expanded to more accurately reflect the physiologicalenvironment of a ligament (e.g. increasing the different types ofmechanical forces) for the in vitro creation of functional equivalentsof native ACL for potential clinical use. These methods are not limitedto the generation of a bioengineered ACL. By applying the appropriatemagnitude and variety of forces experienced in vivo, any type ofligament in the body can be produced ex vivo by the methods of thepresent invention.

1. A fiber construct comprising sericin-extracted silkworm fibroinfibers, wherein the fibers have a diameter of about 20 to about 40 μmand an average ultimate tensile strength of at least about 0.67 N/fiber.2. The fiber construct of claim 1, wherein the fibers are organized inparallel.
 3. The fiber construct of claim 1, wherein the fibers areorganized in a helical, wire rope, twisted, braided, mesh-like or cabledgeometry.
 4. The fiber construct of claim 1, wherein the fibers comprisea coating and/or surface modification that promotes cellular attachmentand/or tissue differentiation and proliferation thereon.
 5. The fiberconstruct of claim 4, wherein said coating and/or surface modificationcomprises an arginine-glycine-aspartate (RGD) peptide.
 6. The fiberconstruct of claim 4, wherein said coating and/or surface modificationcomprises a growth factor.
 7. The fiber construct of claim 1, whereinthe construct comprises a group of at least one to about 1300 fibroinfibers.
 8. The fiber construct of claim 7, wherein the construct furthercomprises at least two groups forming a bundle.
 9. The fiber constructof claim 8, wherein the construct further comprises at least two bundlesforming a strand.
 10. The fiber construct of claim 9, wherein theconstruct further comprises at least two strands forming a cord.
 11. Thefiber construct of claim 7, wherein the group comprises fibroin fibersorganized in a parallel, helical, wire rope, twisted, braided, mesh-likeor cabled geometry.
 12. The fiber construct of claim 8, wherein thebundle comprises groups organized in a parallel, helical, wire rope,twisted, braided, mesh-like or cabled geometry.
 13. The fiber constructof claim 9, wherein the strand comprises bundles organized in aparallel, helical, wire rope, twisted, braided, mesh-like or cabledgeometry.
 14. The fiber construct of claim 10, wherein the cordcomprises strands organized in a parallel, helical, wire rope, twisted,braided, mesh-like or cabled geometry.
 15. The fiber construct of claim1, wherein the construct has an average ultimate tensile strength of atleast about 6.67 N.
 16. The fiber construct of claim 1, furthercomprising cells.
 17. The fiber construct of claim 16, wherein the cellsare selected from the group consisting of stem cells, muscle cells, bonemarrow stromal cells, pluripotent cells, or fibroblast cells.
 18. Amethod for producing a sericin-extracted silk-fibroin fiber constructcomprising: contacting at least one silkworm fibroin fiber with anaqueous solution of Na₂Co₃ and detergent to extract sericin from thefiber.
 19. A method for producing a sericin-extracted silk-fibroin fiberconstruct comprising: a. arranging at least 2 silkworm fibroin fibers toform a group; and b. contacting the group with an aqueous solution ofNa₂Co₃ and detergent to extract sericin from the fibers.
 20. A methodfor producing a sericin-extracted silk-fibroin fiber constructcomprising: a. arranging at least 2 groups of silkworm fibroin fibers toform a bundle; and b. contacting the bundle with an aqueous solution ofNa2Co3 and detergent to extract sericin from the fibers.
 21. A methodfor producing a sericin-extracted silk-fibroin fiber constructcomprising: a. arranging at least 2 bundles of silkworm fibroin fibersto form a strand; and b. contacting the strand with an aqueous solutionof Na2Co3 and detergent to extract sericin from the fibers.
 22. A methodfor producing a sericin-extracted silk-fibroin fiber constructcomprising: a. arranging at least 2 strands of silkworm fibroin fibersto form a cord; and b. contacting the cord with an aqueous solution ofNa2Co3 and detergent to extract sericin from the fibers.
 23. The methodas in one of claims 18-22, wherein the sericin is extracted at atemperature no greater than about 90° C.
 24. The method as in one ofclaims 18-22, further comprising the step of coating the fibroin fiberswith a coating and/or surface modifier that promotes cellular attachmentand/or tissue proliferation on the fibers.
 25. The method of claim 24,wherein said coating and/or surface modifier comprises anarginine-glycine-aspartate (RGD) peptide.
 26. The method of claim 24,wherein said coating and/or surface modifier comprises a growth factor.27. The method of claim 19, wherein said group comprises up to 1300fibroin fibers.
 28. The method of claim 18, further comprising the stepof placing the fiber in a twisted, helical, braided, mesh-like or cabledgeometry.
 29. The method of claim 19, wherein the fibers are organizedin a parallel, helical, wire rope, twisted, braided, mesh-like or cabledgeometry.
 30. The method of claim 20, wherein the groups are organizedin a parallel, helical, wire rope, twisted, braided, mesh-like or cabledgeometry forming a bundle.
 31. The method of claim 21, wherein thebundles are organized in a parallel, helical, wire rope, twisted,braided, mesh-like or cabled geometry forming a strand.
 32. The methodof claim 22, wherein the strands are organized in a in parallel,helical, wire rope, twisted, braided, mesh-like or cabled geometryforming a cord.
 33. The method as in any of claims 18-22, furthercomprising: a. contacting the construct with cells; and b. culturing theconstruct under conditions suitable for cell growth and regeneration.34. The method of claim 33, wherein the cells are stem cells, musclecells, bone marrow stromal cells, pluripotent cells, or fibroblastcells.